The demand for quick and efficient analysis of biochemical and chemical analytes continues to be an area of great interest in the medical and scientific community. For example, from its inception, the Human Genome Project (U.S. Dept. of Energy and Health, Science 262:43-46 (1993)) has called upon existing technologies to provide cost-effective, high-speed and high-throughput nucleic acid sequencing. This was necessary primarily because of the inadequate overall efficiency of traditional slab gel electrophoresis techniques. Alternative technologies, such as capillary array electrophoresis (Huang et al., Anal. Chem. 64:967-972 (1992)), microchannel array electrophoresis (Woolley et al., Proc. Natl. Acad. Sci. (USA) 91:11348-11352 (1994); Woolley et al., Anal. Chem. 67:3676-3680 (1995)), sequencing by hybridization (Drmanac et al., Yugoslav Patent Application 570 (1987); GB 8810400 (1988)), single-molecule sequencing (Davis et al., Genet. Anal. 8:1-7 (1991); Goodwin et al., Nucleosides and Nucleotides 16:543-547 (1997)), and sequencing by mass spectroscopy (Juhasz et al., Anal. Chem. 68:941-946 (1996)) have been explored extensively. Intensive research efforts have also been directed toward improving the individual technologies, such as reaction chemistry, purification, sample injection, separation, detection, and data analysis. Ultimately, integration and automation of the above-described technologies will be critical to the success of the Human Genome Project.
Capillary electrophoresis (CE) is an attractive alternative to conventional slab gel electrophoresis in nucleic acid analysis due to advantages such as high migration speed, high separation efficiency, small sample requirement, and suitability for automation. For example, CE greatly improves nucleic acid sequencing rates compared to conventional slab gel electrophoresis. However, capillary electrophoresis and conventional slab gel technologies have yet to be interfaced with sample processing technologies in a multiplexed system. System integration of CE to robotic arms and conveyer-belts (Mardis et al., BioTechnigues 7:840-850 (1989); Wilson et al., BioTechnigues 6:776-777 (1988)), although workable, suffers from reliability and incompatibility issues because of the many moving parts at the robotic end and the small volumes at the separation and detection end. On the other hand, on-line integrated microfluidics is inherently more compatible with the capillary/microchannel formats. Based on this approach, DNA restriction digestion (Woolley et al., Anal. Chem. 68:4081-4086 (1996)), polymerase chain reaction (Swerdlow et al., Anal. Chem. 69:848-855 (1997)), and cycle sequencing reaction (Tan et al., Anal. Chem. 69:664-674 (1997)), have all been interfaced with capillary/microchannel electrophoresis for sizing. Although these studies successfully coupled sample preparation with separation and detection in a single channel, multiplexing of these elements has not been demonstrated.
It has been previously shown that an on-line integrated microfluidic system from dye-terminator sequencing reaction to called bases is feasible in a single channel (Tan et al., Anal. Chem. 69:664-674 (1997)). This was accomplished by identifying a set of compatible and automatable technologies. In this system, a fused-silica capillary served as the microreactor of cycle-sequencing reaction inside a hot-air thermal cycler. A mini-bore chromatographic column based on size exclusion was used to purify the reaction products. A cross-shaped junction, i.e., a 4-way junction, acted as a multi-functional device for denaturation, pre-concentration, and injection at a high temperature. CE coupled with laser-induced fluorescence was utilized to read the DNA sequence. One of the major obstacles toward multiplexing this approach, however, was the inclusion of four bulky rotary valves. Most of the traditional mechanical valves, whether they are linear valves (gate, globe, diaphragm, or pinch) or rotary valves (ball, plug, butterfly, or shaft), are not suitable for constructing a highly parallel system. Although electro-osmotic flow control represents an available alternative, it is not compatible with the use of a purification column. Furthermore, it is unreliable under changing buffer conditions, which are necessitated by complex manipulations.
Thus, a need exists for an integrated multiplexed on-line system capable of simultaneously analyzing multiple samples. The multiplexed system should provide greater efficacy over conventional procedures with regard to time and further improve the efficiency of separation, purification and detection of analytes contained within the samples.
The present invention provides an advancement in the processing of samples based upon multiplexed microfluidics and capillary array electrophoresis. A system of the invention can process multiple samples and if desired, execute multiple sample manipulation steps, preferably all in a parallel fashion. In one embodiment, the system can contain a plurality of intake capillaries, a chromatographic column array having a plurality of chromatographic columns and a separation capillary array having a plurality of separation capillaries. At least one detector can be integrated into the system to detect analytes eluting from the separation capillaries and/or the chromatographic columns. In another embodiment, the system contains a plurality of intake capillaries each having a reaction portion and a separation capillary array having a plurality of separation capillaries.
A system of the invention typically employs at least one multiplexed freeze-thaw valve assembly (MFTV) that regulates the flow of fluids in the system. Valve assemblies are positioned in a system in a manner that allows sample movement through the integrated components in automated fashion. Sample and fluid movement in the system are typically controlled by a series of valves and pumps that are activated by an electronic signal from a computer. A system of the invention typically contains at least one set of junctions and at least one manifold that permits fluid communication between selected integrated components in the system. A multiplexed system of the invention can advantageously support two sample analysis channels to about one thousand or ore sample analysis channels.
In a first embodiment, the system contains a plurality of intake capillaries, each intake capillary in fluid communication with one of a plurality of first junctions; a chromatographic column array containing a plurality of chromatographic columns having an outlet end, each chromatographic column in fluid communication with one of the plurality of first junctions and one of a plurality of second junctions, the chromatographic column being interposed between the first and second junctions; and a separation capillary array containing a plurality of separation capillaries, each separation capillary in fluid communication with one of the plurality of second junctions and having an outlet end.
In another embodiment, the system contains a plurality of intake capillaries, each intake capillary having a reaction portion for reacting a sample, wherein each intake capillary is in fluid communication with one of a plurality of junctions; and a separation capillary array containing a plurality of separation capillaries, each separation capillary in fluid communication with one of the plurality of junctions and having an outlet end.
The invention further provides at least one multiplexed freeze thaw valve assembly containing a plurality of capillaries, at least a portion of each capillary containing a fluid, and a plurality of heat conductive portions in contact with the fluid-containing portions of the plurality of capillaries; wherein the valve assembly is closed by contacting the heat conductive portions with a cooled liquid to solidify the fluid in the capillaries.
The various methods provided by the present invention include a method for detecting an analyte in at least one sample. Once such method includes the steps of (a) providing a plurality of intake capillaries, each intake capillary having an inlet end and in fluid communication with one of a plurality of first junctions; a chromatographic column array containing a plurality of chromatographic columns having an outlet end, each chromatographic column in fluid communication with one of the plurality of first junctions and one of a plurality of second junctions, the chromatographic column being interposed between the first and second junctions; and a separation capillary array containing a plurality of separation capillaries, each separation capillary in fluid communication with one of the plurality of second junctions and having a distal portion with an outlet end; (b) introducing each of the plurality of samples into the inlet end of an intake capillary; (c) transferring each of the plurality of samples from each intake capillary into a chromatographic column in fluid communication with each of the intake capillaries; (d) chromatographing each of the plurality of samples to yield a plurality of purified sample portions, the purified sample portions comprising at least one detectable analyte; (e) injecting each of the plurality of purified sample portions into a separation capillary in fluid communication with the chromatographic column; (f) separating each of the purified sample portions to yield a plurality of separated sample portions, each of the separated sample portions comprising at least one detectable analyte; and (g) detecting at least one detectable analyte.
Another method includes the steps of: (a) providing a plurality of intake capillaries, having an inlet end in fluid communication with a plurality of junctions, wherein each intake capillary is formed to have a reaction portion; a separation capillary array containing a plurality of separation capillaries, each separation capillary in fluid communication with one of the plurality of junctions and having an outlet end; (b) introducing each of the plurality of samples into inlet end of an intake capillary and transferring each of the plurality of samples into the reaction portion of each intake capillary; (c) reacting each of the transferred samples to yield a reacted sample; (d) injecting each of the plurality of reacted samples into a separation capillary in fluid communication each of the intake capillaries; (e) separating each of the reacted samples to yield a plurality of separated sample portions, each of the separated sample portions comprising at least one detectable analyte; and (f) detecting at least one detectable analyte.
Yet another method provides for sequencing nucleic acids in a plurality of samples that contains the steps of: (a) providing a plurality of intake capillaries, each intake capillary in fluid communication with one of a plurality of first junctions, wherein at least one intake capillary has a reaction portion for reacting a sample; a chromatographic column array containing a plurality of chromatographic columns having an outlet end, each chromatographic column in fluid communication with one of the plurality of first junctions and one of a plurality of second junctions, the chromatographic column being interposed between the first and second junctions; a separation capillary array containing a plurality of separation capillaries, each separation capillary in fluid communication with one of the plurality of second junctions and having an outlet end; a plurality of first freeze-thaw valves for regulating the flow of fluids in the system, each first freeze-thaw valve containing a portion of an intake capillary wherein each of the plurality of first freeze-thaw valves contains a distal freeze thaw valve having a portion of the intake capillary distal to the reaction portion and a proximal freeze-thaw valve having a portion of the intake capillary proximal to the reaction portion; a plurality of first joining capillaries, each first joining capillary interposed between a first junction and a chromatographic column, the first junction being in fluid communication with the intake capillary; a plurality of second freeze-thaw valves for regulating the flow of fluids in the system, each second freeze-thaw containing a portion of a first joining capillary; (b) introducing each of the plurality of samples into the reaction portion of an intake capillary, each sample containing a nucleic acid; (c) reacting each of the plurality of samples in a DNA sequencing reaction to yield a plurality of reacted samples, each reacted sample containing a plurality of detectably labeled nucleic acid fragments having different lengths; (d) transferring each of the plurality of reacted samples from each of the intake capillaries into the chromatographic column in fluid communication with the intake capillary; (e) chromatographing each of the plurality of reacted samples to yield a plurality of purified samples, each purified sample containing a plurality of detectably labeled nucleic acid fragments having different lengths; (f) injecting each of the plurality of purified samples into a separation capillary in fluid communication with the chromatographic column; (g) separating each of the plurality of purified samples to yield, for each purified sample, a plurality of separated detectably labeled nucleic acid fragments having different lengths; (h) detecting the detectably nucleic acid fragments having different lengths; and (i) for each sample, determining the sequence of the nucleic acid.